Dopamine (DA) transmission is involved in important brain functions including locomotor control, neuroendocrine secretion, cognition, emotion, and reward. Dopaminergic signaling is terminated primarily by the re-uptake of the transmitter via a plasma membrane DA transporter (DAT). This protein is also the main target of widely abused psychostimulants, such as cocaine and amphetamine. Recently, our group and others have identified several proteins that interact with DAT and play important roles in targeting, trafficking, and functional regulation of the transporter. Using the yeast mating-based split ubiquitin system, we have now identified the synaptic vesicle protein synaptogyrin-3 (SG3) as such a DAT interacting protein. We have gathered preliminary data suggesting a physical and functional interaction between DAT and SG3. Based on these observations, our central hypothesis is that a physical and functional interaction between SG3 and DAT influences transporter function and ultimately DA homeostasis. This may be due to a link between the plasma membrane DAT-mediated uptake and the vesicular DA storage system, or alternatively, through regulation of DAT targeting, recycling, and/or intrinsic transporter activity. In this application, a combination of biochemical, molecular, and functional approaches in vitro and in vivo will be used with cells in culture, synaptosomal preparations, purified synaptic vesicles, and whole animals to generate a detailed structural, functional, and subcellular description of the DAT/SG3 interaction. Specifically, we will identify the protein residues involved in the DAT/SG3 interaction and examine the specificity of this interaction (Aim 1); investigate the impact of SG3 on DAT function (Aim 2); examine the regulation of the DAT/SG3 interaction (Aim 3); and determine the subcellular location for the DAT/SG3 interaction (Aim 4). The long-term goal of our research program is to understand the mechanisms involved in the regulation of DA homeostasis by DAT and how these mechanisms are altered by psychostimulants. The discovery of novel DAT interacting proteins may suggest novel mechanisms associated with the activity of the transporter, and as a consequence, these mechanisms will have an important impact in the regulation of DA homeostasis. Public Health Statement: Findings from these studies are expected to aid in the advancements of new strategies for intervention in DA-related disorders including drug addiction.